This application is a National Stage application under 35 U.S.C. xc2xa7371 of International Application Serial No. PCT/CN99/00130 filed Aug. 30, 1999 and published in Chinese as WO 00/12721 on Mar. 9, 2000, which claims priority to Chinese Application Serial No. 98111042.8, filed Aug. 31, 1998.
The invention relates to a new polynucletide ,the polypeptide encoded by said polynucleotide, the uses of said polynucleotide and polypeptide, and the methods for preparing same. In particular, the invention relates to a new member of the lysozyme family.
Lysozyme exists ubiquitously in all parts of organisms, including various tissues, organs, and sera; it is especially abundant in egg white. Lysozyme is mainly secreted by the epithelial cell of certain glands and some kinds of leukocyte.
Lysozyme was first reported by Fleming, et al. in 1922. Afterward, lysozyme has been widely studied. A lot of papers concerning its crystal structure, protein catalytic domains, catalytic dynamics, immunology, molecular evolutionary, and so on, have been published. Lysozyme is one of the proteins that are studied most extensively and intensively. However, the study on lysozyme gene is not yet sufficient. Nowadays, only a few lysozyme genes from different species, such as E.coli T4, salmonella P22 phage, bacillus xcfx86 phage and chicken, etc., have been cloned. (1983 J. Mol. Biol. 165. 229-248; 1985 Virology 143, 280-289; 1987 Proc. Natl. Acad. Sci. USA, 77, 5759-5763). The cloning about human lysozyme gene was also reported (1988, Gene 66,223-234).
The main function of lysozyme is to hydrolyze the beta(1-4) glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylgluconic acid (NAG) of the bacterial cell wall. In the organism, lysozyme can act as a nonspecific immune molecule against bacterial infections, and as a digestive enzyme in enteron and some mollusks which live on bacteria. Further, lysozyme has the function of inhibiting tumor growth. Therefore, lysozyme has important applications in both industry and medicine.
One purpose of the invention is to provide a new polynucleotide which encodes a new member of lysozyme gene family. The new human lysozyme is named LYC1.
Another purpose of the invention is to provide a new member of lysozyme protein family, which is named LYC1.
Still another purpose of the invention is to provide a new method for preparing said new human lysozyme by recombinant techniques.
The invention also relates to the uses of said human lysozyme and its coding sequence.
In one aspect, the invention provides an isolated DNA molecule, which comprises a nucleotide sequence encoding a polypeptide having human LYC1 protein activity, wherein said nucleotide sequence shares at least 70% homology to the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4, or said nucleotide sequence can hybridize to the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4 under moderate stringency. Preferably, said nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5. More preferably, the sequence comprises the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4.
Further, the invention provides an isolated LYC1 polypeptide, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 5, its active fragments, and its active derivatives. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 5.
The invention also provides a vector comprising said isolated DNA.
The invention further provides a host cell transformed with said vector.
In another aspect, the invention provides a method for producing a polypeptide with the activity of LYC1 protein, which comprises:
(a) forming an expression vector of LYC1 protein comprising the nucleotide sequence encoding the polypeptide having the activity of LYC1 protein, wherein said nucleotide sequence is operably linked with an expression regulatory sequences, and said nucleotide sequence shares at least 70% homology to the nucleotide sequence of positions 84-530 in SEQ ID NO: 4;
(b) introducing the vector of step (a) into a host cell, thereby forming a recombinant cell of LYC1 protein;
(c) culturing the recombinant cell of step (b) under the conditions suitable for the expression of LYC1 polypeptides;
(d) isolating the polypeptides having the activity of LYC1 protein.
In one embodiment of the present invention, the isolated polynucleotide has a full length of 583 nucleotides, whose detailed sequence is shown in SEQ ID NO: 4. The open reading frame (ORF) locates at nucleotides 84-530.
In the present invention, the term xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpurifiedxe2x80x9d or xe2x80x9csubstantially purexe2x80x9d DNA refers to a DNA or fragment which has been isolated from the sequences which frank it in a naturally occurring state. The term also applied to DNA or DNA fragment which has been isolated from other components naturally accompanying the nucleic acid and from proteins naturally accompanying it in the cell.
In the present invention, the term xe2x80x9cLYC1 protein encoding sequencexe2x80x9d or xe2x80x9cLYC1 polypeptide encoding sequencexe2x80x9d refers to a nucleotide sequence encoding a polypeptide having the activity of LYC1 protein, such as the nucleotide sequence of positions 84-530 in SEQ ID NO: 4 or its degenerate sequence. The degenerate sequences refer to the sequences formed by replacingone or more codons in the ORF of 84-530 in SEQ ID NO: 4 with degenerate codes which encode the same amino acid. Because of the degeneracy of codon, the sequence having a homology as low as about 70% to the sequence of nucleotides 84-530 in SEQ ID NO: 4 can also encode the sequence shown in SEQ ID NO: 5. The term also refers to the nucleotide sequences that hybridize with the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4 under moderate stringency or preferably under high stringency. In addition, the term also refers to the sequences having a homology at least 70%, preferably 80%, more preferably 90% to the nucleotide sequence of nucleotides 84-530 in SEQ ID NO: 4.
The term also refers to variants of the sequence in SEQ ID NO: 4, which are capable of coding for a protein having the same function as human LYC1 protein. These variants includes, but are not limited to: deletions, insertions and/or substitutions of several nucleotides (typically 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) and additions of several nucleotides (typically less than 60, preferably 30, more preferably 10, most preferably 5) at 5xe2x80x2 end and/or 3xe2x80x2 end.
In the present invention, xe2x80x9csubstantially purexe2x80x9d proteins or polypeptides refers to those which occupy at least 20%, preferably at least 50%, more preferably at least 80%, most preferably at least 90% of the total sample material (by wet weight or dry weight). Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, PAGE or HPLC analysis. A substantially purified polypeptides is essentially free of naturally associated components.
In the present invention, the term xe2x80x9cLYC1 polypeptidexe2x80x9d or xe2x80x9cLYC1 proteinxe2x80x9d refers to a polypeptide having the activity of LYC1 protein comprising the amino acid sequence of SEQ ID NO: 5. The term also comprises the variants of said amino acid sequence which have the same function of human lysozyme. These variants include, but are not limited to, deletions, insertions and/or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) at C-terminal and/or N-terminal. For example, the protein function are usually unchanged when an amino residue is substituted by a similar or analogous one. Further, the addition of one or several amino acids at C-terminal and/or N-terminal will not change the function of protein. The term also includes the active fragments and derivatives of LYC1 protein.
The variants of polypeptide include homologous sequences, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes to LYC1 DNA under high or low stringency conditions as well as the polypeptides or proteins retrieved by antisera raised against LYC1 polypeptide. The present invention also provides other polypeptides, e.g., fusion proteins, which include the LYC1 polypeptide or fragments thereof. In addition to substantially full-length polypeptide, the soluble fragments of LYC1 polypeptide are also provided. Generally, these fragments comprise at least 10, typically at least 30, preferably at least 50, more preferably at least 80, most preferably at least 100 consecutive amino acids of human LYC1 polypeptide.
The present invention also provides the analogues of LYC1 protein or polypeptide. Analogues can differ from naturally occurring LYC1 polypeptide by amino acid sequence differences or by modifications which do not affect the sequence, or by both. These polypeptides include genetic variants, both natural and induced. Induced variants can be made by various techniques, e.g., by random mutagenesis using irradiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biologic techniques. Also included are analogues which include residues other than those naturally occurring L-amino acids ( e.g., D-amino acids) or non-naturally occurring or synthetic amino acids (e.g., beta- or gamma-amino acids). It is understood that the polypeptides of the invention are not limited to the representative polypeptides listed hereinabove.
Modifications ( which do not normally alter primary sequence) include in vivo, or in vitro chemical derivation of polypeptides, e.g., acelylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in the further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation (e.g., mammalian glycosylating or deglycosylating enzymes). Also included are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, phosphothronine, as well as sequences which have been modified to improve their resistance to proteolytic degradation or to optimize solubility properties.
The invention also includes antisense sequence of the sequence encoding LYC1 polypeptide. Said antisense sequence can be used to inhibit expression of LYC1 in cells.
The invention also include probes, typically having 8-100, preferably 15-50 consecutive nucleotides. These probes can be used to detect the presence of nucleic acid molecules coding for LYC1 in samples.
The present invention also includes methods for detecting LYC1 nucleotide sequences, which comprises hybridizing said probes to samples, and detecting the binding of the probes. Preferably, the samples are products of PCR amplification. The primers in PCR amplification correspond to coding sequence of LYC1 polypeptide and are located at both ends or in the middle of the coding sequence. In general, the length of the primers is 20 to 50 nucleotides.
A variety of vectors known in the art, such as those commercially available, are useful in the invention.
In the invention, the term xe2x80x9chost cellsxe2x80x9d includes prokaryotic and eukaryotic cells. The common prokaryotic host cells include Escherichi coli, Bacillus subtilis, and so on. The common eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cells are eukaryotic cells, e.g., CHO cells, COS cells, and the like.
In another aspect, the invention also includes antibodies, preferably monoclonal antibodies, which are specific for polypeptides encoded by LYC1 DNA or fragments thereof. By xe2x80x9cspecificityxe2x80x9d is meant an antibody which binds to the LYC1 gene products or a fragments thereof. Preferably, the antibody binds to the LYC1 gene products or a fragments thereof and does not substantially recognize and bind to other antigenically unrelated molecules. Antibodies which bind to LYC1 and block LYC1 protein and those which do not affect the LYC1 function are included in the invention. The invention also includes antibodies which bind to the LYC1 gene product in its unmodified as well as modified form.
The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically-active antibody fragments, e.g., a Fabxe2x80x2 or (Fab)2 fragment, an antibody light chain, an antibody heavy chain, a genetically engineered single chain Fv molecule (Lander, et al., U.S. Pat. No. 4,946,778), or a chimeric antibody, e.g., an antibody which contains the binding specificity of a murine antibody, but the remaining portion of which is of human origin.
The antibodies in the present invention can be prepared by various techniques known to those skilled in the art. For example, purified LYC1 gene products, or itsantigenic fragments can be administrated to animals to induce the production of polyclonal antibodies. Similarly, cells expressing LYC1 or its antigenic fragments can be used to immunize animals to produce antibodies. Antibodies of the invention can be monoclonal antibodies which can be prepared by using hybnrdoma technique (See Kohler, et al., Nature, 256; 495,1975; Kohler, et al., Eur. J. Immunol. 6: 511,1976; Kohler, et al., Eur. J. Immunol. 6: 292, 1976; Hammerling, et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981). Antibodies of the invention comprise those which block LYC1 function and those which do not affect LYC1 function. Antibodies in the invention can be produced by routine immunology techniques and using fragments or functional regions of LYC1 gene product. These fragments and functional regions can be prepared by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies binding to unmodified LYC1 gene product can be produced by immunizing animals with gene products produced by prokaryotic cells (e.g., E. coli); antibodies binding to post-translationally modified forms thereof can be acquired by immunizing animals with gene products produced by eukaryotic cells (e.g., yeast or insect cells).
In one embodiment, the polynucleotide of the invention is 583 bp in full length whose detailed sequence is shown in SEQ ID NO: 4 with the ORF located at positions 84-530. Said polynucleotide was obtained as follows: human brain xcex gt 11 cDNA library (Clontech) was used as a template and PCR was carried out with forward primer A1: 5xe2x80x2-TAAGGAAACCTGGCTGCCCTCTC-3xe2x80x2(SEQ ID NO: 1) and reverse primer B: 5xe2x80x2-CTGAGTGAGGACAGGAGTCTTGG-3xe2x80x2(SEQ ID NO: 2). Then, the PCR amplification product was used as a template and an additional PCR was carried out with primer A2: 5xe2x80x2CCAGGCTCTCAGAGAAGATCAGC-3xe2x80x2(SEQ ID NO: 3) and reverse primer B. Target fragments of 614 bp (product A1B) and 583 bp (product A2B) were obtained in two PCRs, respectively. The sequencing of the PCR product A2B gave the full length cDNA sequence shown in SEQ ID NO: 4.
Homology comparison showed that the nucleotide sequence and the coded protein sequence of the invention shared remarkable homology to other lysozymes from different origins. Therefore, it indicates it is a new member of lysozyme family and has some important functions of the family.
Lysozyme can lyse cells by hydrolyze the beta(1-4) glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylgluconic acid (NAG) of the bacterial cell wall. In the organisms, lysozyme can act as a nonspecific immune molecule against bacterial infections, and as a digestive enzyme in enteron and some mollusks which live on bacteria. Further, lysozyme has the function of inhibiting tumor growth. In 1955, Caselli and Shumacher (Boll Ocul 34:513-533, 1955) reported on the lysozyme-mediated 70% inhibition of neoplastic transformation in cornea of chicken infected by Rous sarcoma virus. Many other experiments indicated that lysozyme participates in the process of tumor diffusion and interacts with phospho- and glucolipid molecule of tumor cells. The inhibition effect on human tumor of lysozyme was reported and patented (1980 Jpn Kokai, Tokkyo Koho 33,409; 1980 Jpn Kokai Tokkyo Koho 33,408). As to the mechanism of lysozyme inhibition on tumor, there are two possibilities: (1) lysozyme directly activates the organism""s immunity functions; (2) lysozyme indirectly enhances the organism""s immune ability (1989 Anticancer Research 9, 583-592).
The invention is further illustrated by the following examples. It is appreciated that these examples are only intended to illustrate the invention, but not to limit the scope of the invention. For the experimental methods in the following examples, they are performed under routine conditions, e.g., those described by Sambrook. et al., in Molecule Clone: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or as instructed by the manufacturers, unless otherwise specified.